As inhibitory molecular species against interaction between biological molecules, a peptide having an α helix secondary structure has attracted attentions mainly from the following three points. First one is that many interactions are mediated by an α helix so that development of an inhibitor making use of this structure as a starting point can be expected; second one is that since the peptide having a helix structure therein has cell membrane permeability with high possibility, a peptide drug targeting an intracellular protein can be created; and third one is that since in spite that it is a peptide, it has acquired stability against protease, it is expected to have a longer half-life in blood than general peptides. In most cases, however, preparation of a peptide simply based on the information of the amino acid sequence constituting the α helix does not lead to acquisition of such advantages. In the case of short-chain peptides, different from proteins having a three-dimensional structure, the circumstance surrounding the helix is exposed to a solvent and therefore the peptides are significantly affected by it so that they cannot keep their structure. With a view to overcoming this problem, there have been many attempts to crosslink amino acid side chains at appropriate positions to each other through a covalent bond in order to support a specific hydrogen bond that constitutes this helix structure, and thereby maintain or fix the helix structure even in an aqueous solution. Many of such approaches however depend all the processes, including peptide synthesis, on chemical synthesis because a chemical catalyst is used to form a covalent bond of an artificial amino acid incorporated in a peptide chain. When special peptides having a fixed α helix secondary structure are required to have physiological activity, peptide drug candidates have so far been found by designing or constructing a low diversity focused library based on the sequence of the α helix site of existing proteins and evaluating the activity of each peptide.
For acquiring a peptidic molecule that binds to a specific target, on the other hand, a method of screening from a random peptide library has been used widely. The most common method is a peptide display method using a phage, but recently, a peptide display method without using biological species such as Escherichia coli has been employed. Described specifically, various in vitro display methods such as ribosome display method and mRNA display method making use of translation are excellent because a high diversity library can be constructed and screened in a tube in a short period of time. The term “in vitro display method” means a system facilitating concentration and amplification (selection) of active species by linking a phenotype and a genotype encoding the sequence thereof through a non-covalent bond or a covalent bond to display the phenotype on the genotype and using a replication system reconstructed in a test tube. The greatest characteristic of this system is that it is conducted without using a prokaryote or eukaryote as a medium so that a high-activity physiological substance can be isolated from a library having great diversity. As a typical comparison example, phage display using Escherichia coli as a replication medium enables selection from a library having diversity as high as 107, while in vitro display enables selection from a library having diversity as high as 1012. Examples of the in vitro display include ribosome display, mRNA display, and RaPID display (unpublished international application PCT/JP2010/68549). As one example, mRNA display will next be described below.
The mRNA display method is a technology of binding a polypeptide to an mRNA which is a template thereof to match the amino acid sequence of the polypeptide to the nucleic acid sequence. By binding puromycin, which is an analogue of the end of acylated tRNA, to the 3′ end of the mRNA via an appropriate linker and adding it to a translation reaction, puromycin penetrates in the A site of ribosome, forms a covalent bond with a peptide during elongation, and as a result, a peptide molecule which is a translation product links to the mRNA via puromycin (Patent Documents 1 to 3, Non-patent Documents 1 and 2).
Thus, the in vitro display enables screening of a peptide library having diversity as high as 1012, but only a peptide library composed only of proteinogenic amino acids has conventionally been constructed because the peptide library is constructed by making use of functions of living body. It is expected that if the diversity of the library is improved by incorporating a novel and non-native function in the structure of amino acids, a peptide library having a novel skeleton capable of stabilizing an α helix secondary structure, which is usually unstable, can be constructed and screened, and that it becomes possible to obtain peptides exhibiting high inhibitory ability, selectivity, stability, and the like which naturally-occurring simple peptide chains cannot achieve.
With recent development in technology called “genetic code expansion” or “reprogramming of genetic code”, it has actually become possible to produce and screen a peptide library having special amino acids by using various display methods such as phage display.
In genetic code expansion, it becomes possible to synthesize proteins or peptides containing a special amino acid by making use of stop codons or artificial four-base codons which are not used for assignment of an amino acid in a natural translation system and allocating these codons to the special amino acid. The number of usable special amino acids is however limited because the number of stop codons or usable four-base codons is limited (substantially, three or less special amino acids).
Studies on peptides having a crosslinked structure introduced therein have been made extensively in order to stabilize the α helix. There is a report on the method making use of an amide bond, a disulfide bond, and alkene formation through a ring-closing metathesis reaction (Non-patent Document 3). Introduction of these covalent bonds induces a helix structure, but this method has generally a problem in in vivo stability. Described specifically, the amide structure or disulfide structure is easily cleaved by protease or under the reduction conditions so that using such a peptide as an inhibitor is disadvantageous. In order to overcome this disadvantage, a method using alkene formation is developed. The peptide having a crosslinked structure formed by this method is found to have activity in vivo (Non-patent Document 4). A physiologically active peptide having a crosslinked structure by using alkene formation is under development.
The development however needs tremendous labor because after construction of a focused library from known amino acid sequences by chemical synthesis, the position of the crosslinked structure not depriving physiological activity must be examined precisely. In addition, the peptide sequence obtained from the thus-designed focused library as described above cannot always be the best sequence. Unfortunately, it is difficult to apply the crosslinked reaction making use of alkene formation to the in vitro display method because it uses a chemical catalyst. Described specifically, the alkene formation should be conducted in the presence of factors such as ribosome, protein, and ATP necessary for the translation reaction. They may presumably deteriorate the translation reaction efficiency, making it difficult to construct a high-quality library. In order to discover a physiologically active special peptide having a fixed α helix structure and useful for wider-range of targets, development of a crosslinking method that can be applied to the in vitro display method is indispensable. What is necessary for the crosslinking method is that the crosslinking reaction occurs rapidly and at high selectivity under the conditions in a translation system and the crosslinked structure thus formed is not easily degraded in vivo.